International Journal of Molecular Sciences Review Plant-Derived Inhibitors of AHL-Mediated Quorum Sensing in Bacteria: Modes of Action Dmitry Deryabin *, Anna Galadzhieva, Dianna Kosyan and Galimjan Duskaev Federal Scientific Center of Biological Systems and Agrotechnologies of RAS, Orenburg 460000, Russia; [email protected] (A.G.); [email protected] (D.K.); [email protected] (G.D.) * Correspondence: [email protected]; Tel.: +7-903-2213963; Fax: +7-353-2434641 Received: 7 October 2019; Accepted: 6 November 2019; Published: 8 November 2019 Abstract: Numerous gram-negative phytopathogenic and zoopathogenic bacteria utilise acylated homoserine lactone (AHL) in communication systems, referred to as quorum sensing (QS), for induction of virulence factors and biofilm development. This phenomenon positions AHL-mediated QS as an attractive target for anti-infective therapy. This review focused on the most significant groups of plant-derived QS inhibitors and well-studied individual compounds for which in silico, in vitro and in vivo studies provide substantial knowledge about their modes of anti-QS activity. The current data about sulfur-containing compounds, monoterpenes and monoterpenoids, phenylpropanoids, benzoic acid derivatives, diarylheptanoids, coumarins, flavonoids and tannins were summarized; their plant sources, anti-QS effects and bioactivity mechanisms have also been summarized and discussed. Three variants of plant-derived molecules anti-QS strategies are proposed: (i) specific, via binding with LuxI-type AHL synthases and/or LuxR-type AHL receptor proteins, which have been shown for terpenes (carvacrol and l-carvone), phenylpropanoids (cinnamaldehyde and eugenol), flavonoid quercetin and ellagitannins; (ii) non-specific, by affecting the QS-related intracellular regulatory pathways by lowering regulatory small RNA expression (sulphur-containing compounds ajoene and iberin) or c-di-GMP metabolism reduction (coumarin); and (iii) indirect, via alteration of metabolic pathways involved in QS-dependent processes (vanillic acid and curcumin). Keywords: quorum sensing (QS); AHL-mediated QS; bacterial virulence; biofilm; natural compounds; phytochemicals; quorum sensing inhibitors 1. Introduction Quorum sensing (QS) is a cell–cell communication system that is ubiquitously used in microbial communities to monitor their population density and adapt to external environment. Firstly, QS was named and discovered in the marine bacterium Vibrio fischeri (now Aliivibrio fischeri)[1], where it regulates bioluminescence development in symbiotic “light” organs of squids from the genera Euprymna and Sepiola. This phenomenon involves LuxI synthase, which produces small diffusible signal molecules—acylated homoserine lactones (AHLs)—that accumulate in the environment. Upon reaching a high concentration, AHLs bind with LuxR activator protein and induce lux-operon transcription in a synchronous manner. Many other Gram-negative proteobacteria that belong to α, β and γ subdivisions use similar LuxI-type synthases and LuxR-type activator proteins. They utilise AHL-dependent gene expression mechanisms to perform processes that are not effective at low cell density but very useful for the microbial community at high cell density [2]. Notably, the virulence factors (toxins, proteases and immune-evasion factors) in many zoopathogenic and phytopathogenic bacteria, including Pseudomonas spp., Acinetobacter spp., and Burkholderia spp., are reportedly mediated by AHL. This fact positions QS as an attractive novel target for anti-infective therapy [3]. Another QS-related process is biofilm formation, in which Int. J. Mol. Sci. 2019, 20, 5588; doi:10.3390/ijms20225588 www.mdpi.com/journal/ijms Int. J. Mol. Sci. 2019, 20, 5588 2 of 22 bacterial cells attach to surfaces and envelop themselves in a secreted exopolymeric matrix. In contrast to bioluminescence, virulence factor biosynthesis and some other features, biofilm formation is not strictly switched on by AHLs. However, these phenomena are evolutionarily related [4], and some mechanisms of matrix development are under QS control [5]. Because QS interference aims to reduce virulence and inhibit biofilms but not necessarily kill bacteria, it probably does not exert selective pressure and is less likely to select for resistant strains compared to using conventional antibiotics. Despite that the current list of cell-to-cell communication systems has significantly expanded, a variety of novel autoinducers have been identified, and that hierarchical or parallel QS networks that integrate several regulatory signals and receptors have been described [6], AHL-mediated systems remain the most attractive target for antivirulence therapy in several Gram-negative bacterial families [7]. Over the past 20 years, numerous artificial strategies have been proposed to combat AHL-mediated QS, including suppressing LuxI-type synthases, autoinducer degradation by enzymes (such as lactonases and acylases) or their sorption and sequestration in the environment, LuxR-type receptor antagonism and suppression of QS-activated genes [8]. However, the biopharmaceutical perspectives of these methods are still not completely understood. An alternative approach is the search for natural compounds that show anti-QS activity. In particular, because higher plants co-evolved with the microbial environment and are constantly exposed to bacterial infections, it is logical to expect that these organisms developed have sophisticated chemical mechanisms to combat pathogens, including QS suppression [9]. The aim of this review was to summarize current data about the most significant groups of plant-derived inhibitors of AHL-mediated QS in bacteria with focus on the well-studied individual compounds which in silico, in vitro and in vivo studies taken together allow us to obtain the most complete knowledge about their modes of anti-QS activity. 2. Methodology for the Search and Study of Plant-Derived QS Inhibitors The first step for screening of anti-QS activity is based on analyses of medicinal plants ethnobotanical descriptions. These species are known for their use in the treatment and prevention of bacterial infections in traditional medical practice [10]. Other higher plants that are potential natural QS inhibitor sources are some vegetables, fruits, berries, grains and spices [11]. These species are part of the human diet and may prevent the colonisation and invasion of bacterial pathogens. The selected plant material is dried and treated with water, ethanol or ethyl acetate, which allows the most complete extraction of chemical compounds with different degrees of polarity [12]. The preliminary screening of the obtained extracts includes determination of their direct antibacterial effects, including the use of agar diffusion or micro-broth dilution assays [12–14]. For further studies, concentrations (dilutions) lower than the minimal inhibitory concentration (sub-MIC) only are used [14,15]. The second stage is aimed at screening plant extracts to determine biological activity against bacterial species that use AHL-mediated QS mechanisms for functional differentiation and biofilm formation. Apply the same methods as in the preliminary stage: diffusion of plant extract into agar; followed by measuring the area of suppression of pigments, the production of which depends on QS (any IQS activity is evident by the formation of a colourless, opaque, but visible halo around the well, due to a loss of pigmentation [12–14]); and the method of microbulion dilution (pigment is determined quantitatively by measuring the optical density using a spectrophotometer [12,13,15]). The first method is qualitative and semi-quantitative, it allows the identification of QS inhibitors among plant extracts and to determine the preliminary degree of activity for the subsequent selection of concentrations to work with the dilution method, which is quantitative and allows the ranking of plant extracts by activity. Two types of bioassays can be used for these studies. The first is based on AHL biosensors that have a functional LuxR-type protein but lack the LuxI-type synthase. The most popular biosensor is Chromobacterium violaceum 026 (NCTC 13278), a double mini-Tn5 mutant with insertion of this Int. J. Mol. Sci. 2019, 20, 5588 3 of 22 transposon in the cviI (luxI-type) gene, which synthesises the violet pigment violacein in response to C6-AHL autoinducer concentrations [13]. Other AHL biosensors are recombinant bacteria that carry plasmids with a LuxR-type protein-encoding gene and a QS-controlled promoter fused to the “reporter” genes, including lux- or gfp-operons [16,17]. The promoter activity in these strains depends on the presence of exogenous AHL. Thus, on one hand, the violacein production and bioluminescence level quantify the QS autoinducer presence in the environment; on the other hand, these bioassays allows the evaluation of the anti-QS activity of plant extracts at controlled AHL concentrations that induce violacein biosynthesis and bioluminescence development. Notably, these assays do not implicate the anti-QS properties that target AHL synthesis that may be studied using wild-type sensor strains. One example of an AHL-producing bacterium that is often used for anti-QS activity evaluation is C. violaceum ATCC 31532 [13], which synthesises C6-AHL and represents the initial strain for C. violaceum 026. Another example is Pseudomonas aeruginosa PAO1 (ATCC 15692) [18], which exploits the hierarchical network, including two AHL molecules—3-oxo-C12-AHL and C4-AHL—that